Many compounds are chiral, and it is often desirable to obtain a substantially pure enantiomer from the racemate that is usually available, by conventional chemical synthesis. Such chiral compounds include a number of 2-arylpropionic acids that are well known as anti-inflammatory agents. Examples include ibuprofen, naproxen and ketoprofen. It is now well established that the major therapeutic activity of each resides in the (S)-enantiomer.
Several methods for obtaining the (S)-enantiomer, free of contaminating (R)-enantiomer, are known. These include asymmetric chemical synthesis and chemical resolution such as the stoichiometric crystallisation of diastereomeric salts formed with various chiral amines.
An alternative approach is biocatalytic, using a biocatalyst to selectively hydrolyse an ester of the 2-arylpropionic acid. If a biocatalyst with the correct stereo-specificity can be identified, a reaction mixture containing the unreacted ester of one enantiomer plus the acid product of the other enantiomer can be obtained. Separation and recovery of the product are then relatively facile. Unreacted ester can be racemised and reused in a further reaction, thereby ensuring almost complete conversion of the racemic substrate to the required single enantiomer product.
EP-A-0227078 describes the use of several extracellular, commercially-available microbial lipases in such biocatalytic resolutions. In general, however, large amounts of enzyme were required and the reaction took 2-6 days. Such reactions would therefore be expensive to operate. The best enzyme powder identified was that from Candida cylindraceae (also known as Candida ruposa); however, in EP-A-0407033, it was shown that this preparation, as well as having low activity, contained more than one enzyme with esterase activity. Further, in order to obtain ketoprofen with a high ee (enantiomeric excess), it was necessary to purify the preparation.
EP-A-0233656 describes the isolation and cloning of an esterase gene from Bacillus thai. This enzyme is shown to selectively hydrolyse the ethyl and methyl esters of both naproxen and ibuprofen to give the (S)-acid of respective compounds. It is also shown that cloning of the enzyme resulted in a preparation giving a higher ee product, as a result of minimising other enzyme side-activities.
WO-A-9323547 describes a number of strains which also produce an esterase which selectively hydrolyses naproxen esters. The best strain found was Zopfiella latipes, the esterase from which had been cloned.
WO-A-9304189 describes an organism, Trichosporon sp, that is able to selectively hydrolyse the (S)-enantiomer of ethyl ketoprofen to give an (S)-acid product having an ee of &gt;90%. The enzyme that carries out this biotransformation is intracellular. As stable cell-free preparations have proved difficult to obtain, it is difficult to increase biocatalytic activities by gene cloning or classical mutagenesis.
For economic biotransformation, it is important that cloning techniques should be available, to help reduce enzyme costs. In EP-A-0233656, the relative specific activity of the esterase in the wild Bacillus isolate was very low; thus, even after cloning and hyper-expression of the enzyme, biocatalyst costs are still likely to be significant.
An object behind the present invention has been to obtain a biocatalyst which has good activity against esters, which gives good ee acid product, and which would be suitable for cloning and hyper-expression, e.g. in E. coli, to enable biocatalyst costs to be kept to a minimum.